Painter G R, Diliberto E J, Knoth J
Division of Organic Chemistry, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Proc Natl Acad Sci U S A. 1989 Apr;86(7):2239-42. doi: 10.1073/pnas.86.7.2239.
31P NMR was used to resolve and determine the relative quantity and mobility of ATP in the cytosolic and vesicular compartments of isolated adrenomedullary chromaffin cells. The cells were cultured on microcarrier beads and superfused with an oxygenated medium--thereby permitting dense suspensions of viable cells to be maintained in the NMR probe for extended time periods. Under these conditions, distinct 31P signals could be seen for ATP within the vesicular and the cytosolic pools. Comparison of the integrated areas of the beta-phosphate resonances from the two ATP pools indicated 77% of the endogenous ATP was in the vesicular pool. From this observation and the assumption that the concentration of ATP in the vesicle is 87.5 mM, the concentration of ATP calculated to be in the cytoplasmic pool was approximately 4 mM. The pH in the vesicle determined from the chemical shift of the gamma-phosphate resonance of vesicular ATP was 5.84 +/- 0.17 (n = 6), slightly higher than the intragranular pH measured in hypoxic cells (5.57 +/- 0.15, n = 8). Spin-lattice relaxation times of ATP 31P resonances in the vesicular pool were from 12 to 14 times shorter than the ATP resonances in the cytosol, corresponding to a decrease in molecular mobility due to incorporation of ATP within a catecholamine-storage complex.
采用³¹P核磁共振技术解析并确定分离的肾上腺髓质嗜铬细胞胞质和囊泡区室中ATP的相对含量及流动性。细胞培养在微载体珠上，并用含氧培养基进行灌流，从而使活细胞的密集悬浮液能够在核磁共振探头中长时间维持。在这些条件下，囊泡和胞质库中的ATP可观察到明显的³¹P信号。比较两个ATP库中β-磷酸共振的积分面积表明，77%的内源性ATP存在于囊泡库中。根据这一观察结果以及假设囊泡中ATP的浓度为87.5 mM，计算得出细胞质库中ATP的浓度约为4 mM。根据囊泡ATP的γ-磷酸共振化学位移确定的囊泡内pH值为5.84±0.17（n = 6），略高于缺氧细胞中测得的颗粒内pH值（5.57±0.15，n = 8）。囊泡库中ATP的³¹P共振的自旋晶格弛豫时间比胞质溶胶中的ATP共振短12至14倍，这对应于由于ATP掺入儿茶酚胺储存复合物中导致分子流动性降低。
